Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults, and its incidence increases with age. The minority of patients are cured by current therapy and the majority relapse within 5 years despite continuous treatment. Cancer stem cells (CSCs), which have the capacity for indefinite self-renewal, may be essential for tumor initiation and relapse. New targets are needed to efficiently inhibit CSC activity. Most recently, we identified the immune inhibitory receptors, human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and its mouse ortholog paired Ig-like receptor (PirB), as receptors for Angiopoietin-like proteins (Angptls). Previous studies showed that several Angptls promote cancer development, whereas immune inhibitory receptors suppress the immune activation to support cancer growth. Our preliminary research showed that LILRB2 level is significantly elevated in the M5 subtype of human AML. Angptls bind to LILRB2 and to PirB and induce activation of tyrosine phosphotase SHP-2 and calcium/calmodulin-dependent protein kinase 4 (CAMK4). A deficiency of PirB in two mouse AML models resulted in increased differentiation and decreased self-renewal of leukemia stem cells. Concordantly, the knockdown of LILRB2 in two human leukemia cell lines largely blocked leukemia development in xenografted mice. These results indicate that LILRB2 and PirB support leukemia development. Therefore, our study revealed unexpected functional significance of classical immune inhibitory receptors in the maintenance of stemness of cancer stem cells. We hypothesize that Angptls bind LILRB2 and PirB to inhibit differentiation and promote self-renewal through activating SHP-2 and CAMK4. We propose three aims to test this hypothesis. In Aim 1, we will determine the role of LILRB2 and its ligand binding in regulation of primary human AML-SC activity. In Aim 2, we will dissect the Angptl receptor initiated signaling pathway in AML-SCs, using gain-of-function and loss-of- function approaches to illuminate the functional significance of the SHP-2 signaling and CAMK4 signaling in regulation of self-renewal and differentiation of AML-SCs. In Aim 3, we will investigate how the downstream targets of the Angptl receptor-mediated signaling regulate AML-SC activity. To our knowledge, this is the first study of the molecular mechanism of these inhibitory receptors in the maintenance of stemness of cancer stem cells and progenitors. The proposed study will determine the functional significance of the Angptl receptor in human AML and identify the molecular determinants of the Angptl receptor-mediated signaling in control of AML-SC activity. It will likely open a new research front to study the role of inhibitory receptors in stem cells and cancer. Moreover, the understanding of the ligand-receptor binding, the signaling events, and the downstream effectors in this newly identified pathway will directly guide the development of new therapeutic strategies for human AML.